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Rho GTPase-independent regulation of mitotic progression by the RhoGEF Net1.

Menon S, Oh W, Carr HS, Frost JA - Mol. Biol. Cell (2013)

Bottom Line: Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation.The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1.These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX 77008, USA.

ABSTRACT
Neuroepithelial transforming gene 1 (Net1) is a RhoA-subfamily-specific guanine nucleotide exchange factor that is overexpressed in multiple human cancers and is required for proliferation. Molecular mechanisms underlying its role in cell proliferation are unknown. Here we show that overexpression or knockdown of Net1 causes mitotic defects. Net1 is required for chromosome congression during metaphase and generation of stable kinetochore microtubule attachments. Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation. The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1. We also observe that Net1 expression is required for centrosomal activation of p21-activated kinase and its downstream kinase Aurora A, which are critical regulators of centrosome maturation and spindle assembly. These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

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Net1 depletion interferes with mitotic progression. HeLa cells were transfected with control or Net1-specific siRNAs. One day later the cells were retransfected with a plasmid expressing mCherry-H2B. Live cell imaging was performed 2 d after that to monitor mitotic progression. (A) Representative still frames from control siRNA–transfected cells. The time elapsed from nuclear envelope breakdown is shown in the lower right-hand corner (minutes). (B) Mitotic progression in two different Net1 siRNA–transfected cells. Middle, arrows indicate lagging chromosomes. Bar, 10 μm. (C) Quantification of time required to progress through different phases of mitosis in control and Net1 siRNA–transfected cells. Errors are SEM. Statistical significance was determined by Student's t test; **p < 0.01; ***p < 0.001.
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Figure 3: Net1 depletion interferes with mitotic progression. HeLa cells were transfected with control or Net1-specific siRNAs. One day later the cells were retransfected with a plasmid expressing mCherry-H2B. Live cell imaging was performed 2 d after that to monitor mitotic progression. (A) Representative still frames from control siRNA–transfected cells. The time elapsed from nuclear envelope breakdown is shown in the lower right-hand corner (minutes). (B) Mitotic progression in two different Net1 siRNA–transfected cells. Middle, arrows indicate lagging chromosomes. Bar, 10 μm. (C) Quantification of time required to progress through different phases of mitosis in control and Net1 siRNA–transfected cells. Errors are SEM. Statistical significance was determined by Student's t test; **p < 0.01; ***p < 0.001.

Mentions: To evaluate the effect of Net1 depletion on mitotic progression, we performed live imaging of HeLa cells transfected with control and Net1 small interfering RNAs (siRNAs), plus an expression plasmid for histone H2B-mCherry to visualize chromatin. Live cell imaging confirmed that loss of Net1 expression had a profound effect on the ability of cells to progress through mitosis. Control siRNA–transfected cells completed mitosis in an average of 45 min, in agreement with previously published results in this cell type (Figure 3A; Yoshizaki et al., 2003; Oceguera-Yanez et al., 2005; Schmidt et al., 2007). However, ∼60% of the Net1-depleted cells never completed mitosis during the 4-h imaging period. The remaining cells were significantly delayed in mitotic progression, requiring 90–160 min to complete cell division (Figure 3, B and C). In arrested cells it was apparent that some of the chromatin never properly aligned to the metaphase plate. This type of defect would be expected to engage the spindle assembly checkpoint and prevent cells from entering anaphase. In the cells that completed mitosis, quantification revealed a significant delay in progression from prometaphase to metaphase, as well as from metaphase to anaphase. There was no discernible delay in progression from anaphase to telophase (Figure 3C). Of importance, the cells that progressed through anaphase often contained one or more lagging chromosomes, suggesting that spindle attachments were aberrant. Thus these results indicate that Net1 expression is required for cells to align all of their chromosomes on the metaphase plate and faithfully progress through mitosis. These data also explain why we observed so many Net1-knockdown cells with misshapen nuclei or micronuclei, since these phenotypes typically occur after errors in chromatin separation during mitosis.


Rho GTPase-independent regulation of mitotic progression by the RhoGEF Net1.

Menon S, Oh W, Carr HS, Frost JA - Mol. Biol. Cell (2013)

Net1 depletion interferes with mitotic progression. HeLa cells were transfected with control or Net1-specific siRNAs. One day later the cells were retransfected with a plasmid expressing mCherry-H2B. Live cell imaging was performed 2 d after that to monitor mitotic progression. (A) Representative still frames from control siRNA–transfected cells. The time elapsed from nuclear envelope breakdown is shown in the lower right-hand corner (minutes). (B) Mitotic progression in two different Net1 siRNA–transfected cells. Middle, arrows indicate lagging chromosomes. Bar, 10 μm. (C) Quantification of time required to progress through different phases of mitosis in control and Net1 siRNA–transfected cells. Errors are SEM. Statistical significance was determined by Student's t test; **p < 0.01; ***p < 0.001.
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Related In: Results  -  Collection

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Figure 3: Net1 depletion interferes with mitotic progression. HeLa cells were transfected with control or Net1-specific siRNAs. One day later the cells were retransfected with a plasmid expressing mCherry-H2B. Live cell imaging was performed 2 d after that to monitor mitotic progression. (A) Representative still frames from control siRNA–transfected cells. The time elapsed from nuclear envelope breakdown is shown in the lower right-hand corner (minutes). (B) Mitotic progression in two different Net1 siRNA–transfected cells. Middle, arrows indicate lagging chromosomes. Bar, 10 μm. (C) Quantification of time required to progress through different phases of mitosis in control and Net1 siRNA–transfected cells. Errors are SEM. Statistical significance was determined by Student's t test; **p < 0.01; ***p < 0.001.
Mentions: To evaluate the effect of Net1 depletion on mitotic progression, we performed live imaging of HeLa cells transfected with control and Net1 small interfering RNAs (siRNAs), plus an expression plasmid for histone H2B-mCherry to visualize chromatin. Live cell imaging confirmed that loss of Net1 expression had a profound effect on the ability of cells to progress through mitosis. Control siRNA–transfected cells completed mitosis in an average of 45 min, in agreement with previously published results in this cell type (Figure 3A; Yoshizaki et al., 2003; Oceguera-Yanez et al., 2005; Schmidt et al., 2007). However, ∼60% of the Net1-depleted cells never completed mitosis during the 4-h imaging period. The remaining cells were significantly delayed in mitotic progression, requiring 90–160 min to complete cell division (Figure 3, B and C). In arrested cells it was apparent that some of the chromatin never properly aligned to the metaphase plate. This type of defect would be expected to engage the spindle assembly checkpoint and prevent cells from entering anaphase. In the cells that completed mitosis, quantification revealed a significant delay in progression from prometaphase to metaphase, as well as from metaphase to anaphase. There was no discernible delay in progression from anaphase to telophase (Figure 3C). Of importance, the cells that progressed through anaphase often contained one or more lagging chromosomes, suggesting that spindle attachments were aberrant. Thus these results indicate that Net1 expression is required for cells to align all of their chromosomes on the metaphase plate and faithfully progress through mitosis. These data also explain why we observed so many Net1-knockdown cells with misshapen nuclei or micronuclei, since these phenotypes typically occur after errors in chromatin separation during mitosis.

Bottom Line: Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation.The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1.These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX 77008, USA.

ABSTRACT
Neuroepithelial transforming gene 1 (Net1) is a RhoA-subfamily-specific guanine nucleotide exchange factor that is overexpressed in multiple human cancers and is required for proliferation. Molecular mechanisms underlying its role in cell proliferation are unknown. Here we show that overexpression or knockdown of Net1 causes mitotic defects. Net1 is required for chromosome congression during metaphase and generation of stable kinetochore microtubule attachments. Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation. The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1. We also observe that Net1 expression is required for centrosomal activation of p21-activated kinase and its downstream kinase Aurora A, which are critical regulators of centrosome maturation and spindle assembly. These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

Show MeSH
Related in: MedlinePlus